The present invention relates to a porous substrate capable of adsorbing protein and a process for producing the porous substrate. More particularly, the present invention relates to a porous substrate capable of adsorbing protein, which comprises a cross-linked copolymer having methylol groups, produced by subjecting a monomeric mixture of at least one monomer selected from styrene, alpha-methylstyrene and chloromethylstyrenes and a cross-linking agent copolymerizable with the monomer to suspension copolymerization in an aqueous solution of a water-soluble high polymeric substance in the presence of a pore-regulator and introducing methylol groups into the resultant cross-linked copolymer. The outer surface of the substrate is hydrophilic due to the water-soluble high polymeric substance which has been present in the polymerization and attached to the outer surface of the substrate, and the surface of the pores in the substrate is hydrophobic or less hydrophilic than the outer surface of the substrate.
Recently, the importance of a substrate capable of specifically interacting with a substance in a living body has been increased. Namely, as a result of frequent utilization of the high-speed liquid chromatography as a means of analyzing such a substance, the development of a substrate which is to be used in the high-speed liquid chromatography and provided with an excellent specific interaction with such a substance has been demanded. Particularly, for obtaining various informations from the chromatograph of a substance in a living body, which is relatively easily available such as urine, serum, etc., the substrate for use in high-speed liquid chromatography of such a substance is regarded as important. In addition, in the case where such a substrate can be dispersed in the urine and serum and has an interactive function with a specified substance, the substrate can be applied to affinity chromatography, and further, such a substrate is can be applied to antigen-antibody reaction.
Hitherto, as a process for producing cross-linked polymer of styrene or a derivative of styrene, a process of copolymerising styrene and a cross-linking agent copolymerizable with styrene in a state of suspension in water in the presence of a pore-regulator has been known, and for instance, process for producing copolymer of styrene and divinylbenzene (as a cross-linking agent) has been precisely described in J. Polymer Sci., Part A-2, 835(1964). In the described process, wherein a water-soluble high polymeric substance is used as the major suspension stabilizer (suspending agent), although it is possible to produce fine particles of the cross-linked copolymer, an excessive amount of the water-soluble polymeric substance used in the polymerization in suspension adheres onto the surface of the fine particles of the cross-linked copolymer of styrene, and the thus adhered water-soluble high polymeric substance cannot be completely removed from the surface of the fine particles of the cross-linked copolymer even after repeated washing. In the case of applying the thus produced fine particles of the cross-linked copolymer as a substrate, for instance, in the gel-permeation chromatography (hereinafter referred to as GPC) combined with an organic solvent, the pressure necessary for carrying out the chromatography is larger than that in the ordinary case because of the viscous drag phenemenon occuring between the layer of the water-soluble high polymeric substance adhered onto the surface of the fine particles of the cross-linked copolymer and the fluid used in GPC, and together with the occurrence of a certain interaction of the water-soluble high polymeric substance with the component of the specimen to be analized, the phenomenon precluded the use of such a substrate in the liquid chromatography.
On the other hand, in cases where a phosphate such as calcium phosphate, magnesium phosphate, etc. is used as the suspension stabilizer (suspending agent) instead of the water-soluble high polymeric substance, the phosphate adhered to the surface of the fine particles of the cross-linked copolymer is easily removable by washing the particles with an acid, etc., and clean particles of the cross-linked copolymer of styrene can be obtained. However, because of the hydrophobic property of the surface of the thus obtained fine particles of the cross-linked copolymer, the particles cannot be dispersed in water, various aqueous buffer solutions, serum and urine, and cannot absorb proteins within the pores thereof and accordingly, the process cannot solve the problem of offering a substrate having the adsorbency toward protein which is the objective of the present invention.
As a result of studies of the present inventors, it has been found that by chemically introducing methylol groups into the dried porous microspherical particles of the cross-linked copolymer obtained by subjecting a monomeric mixture of at least one monomer selected from the group consisting of styrene, alpha-methylstyrene and chloromethylstyrenes and a cross-linking agent copolymerizable with the monomer to suspension copolymerization in an aqueous solution of a water-soluble high polymeric substance as a suspension stabilizer (suspending agent) in the presence of a pore-regulator, a substrate having its outer surface rich in hydrophilicity and the pores of which the surface is partly hydrophilic, however, richer in hydrophobicity than the outer surface is obtained, and that such a substrate is not only favorably dispersible in serum, urine, etc. but also is able to take proteins into the pores thereof and adsorb the thus taken protein therein, and the present invention has been accomplished based on these findings.